(United States Fifth Circuit) - Held that insurers that paid a claim arising from the failure of a floating oil-drilling platform could not proceed with a subrogation claim against an engineering firm that helped secure the platform to the ocean floor. Also addressed an arbitrability issue. Affirmed a dismissal.

(California Court of Appeal) - Upheld San Francisco's decision to order a prime contractor on a public works project to replace a subcontractor. Reversed the trial court.

(United States Fifth Circuit) - Held that insurers that paid a claim arising from the failure of a floating oil-drilling platform could not proceed with a subrogation claim against an engineering firm that helped secure the platform to the ocean floor. Also addressed an arbitrability issue. Affirmed a dismissal.

ASM Completes Its 150th Film Score And Retains Jonathan Firstenberg As North American Rep

"Whiskey And Cigarettes" Country Radio Show, In Association With The European Academy Of Country Music (EACM) Has Announced Their Top 30 Of 2018 Year-end Chart. The Show Is Syndicated On More Than 25

A new study demonstrates the role of culture in welcoming children with Down syndrome.

For years, I have read news headlines in Christian and secular outlets, from conservatives and liberals alike, stating that “90% of babies with Down syndrome are aborted.” As I have argued before, and as a new study demonstrates, it’s just not true. Correcting this common misconception matters both in providing accurate information to pregnant women and in normalizing the experience of receiving a child with Down syndrome.

Last week, the American Journal of Medical Geneticspublished a study about the birth rates of babies with Down syndrome in the United States. The study estimates live births of babies with Down syndrome from 1900-2010, with a focus upon the effect of prenatal screening programs within the United States on the population of babies with Down syndrome. After factoring in the expected number of live births, miscarriages, and stillbirths, it then posits a “reduction rate.” This reduction rate demonstrates the number of babies with Down syndrome that are not born as a result of selective abortion.

To be specific, in 2006-2010 theses researchers estimate around 5,300 babies were born with Down syndrome annually. During this same time period, approximately 3,100 babies with Down syndrome were selectively aborted each year. Around 800 of those aborted babies would have died before birth, so without selective abortion, the researchers estimate there would be around 7,600 live births with Down syndrome. The reduction rate of babies with Down syndrome in the United States in 2010 was around 30 percent. In other words, without selective abortion, the number of babies born with Down syndrome in recent years would have been about 30 percent higher than it actually has been.

So why ...

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(United States Fifth Circuit) - Held that insurers that paid a claim arising from the failure of a floating oil-drilling platform could not proceed with a subrogation claim against an engineering firm that helped secure the platform to the ocean floor. Also addressed an arbitrability issue. Affirmed a dismissal.

Seven Seas Music Will Now Represent The Re-recorded 11-track Album For Placements In Film Trailers, Advertising, Video Games, And More.

Construction of load-carrying geosynthetic-reinforced bridge abutments are surging worldwide. The design directly supports the load of bridge girders without the need of deep foundation. TRB is hosting a webinar on Tuesday, May 26, 2020 from 2:00 to 4:00 PM Eastern that will provide an overview, comparison, and discussion of methodologies used in the design of such bridge abutments. They have the potential of alleviating the “bump at the bridge,” expediting construction, and requiring comparatively smal...

Platform.sh removes a major pain point for developers: having to invest time in managing servers, virtual machines, or containers. Instead, Platform.sh enables developers to focus 100% of their time on their code. Since the beginning, Platform.sh has provided instant cloning capability, so dev teams can work on perfect copies of their production sites in the cloud for every Git branch. Now, in partnership with Lando, we’re extending that capability to the desktop.

Ничто так не подчёркивает важность достоверных новостей, как кризис. Однако пандемия сovid-19 переворачивает вверх дном журналистику и меняет условия труда. Нельзя недооценивать возникающий в результате стресс для психического здоровья работников СМИ.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration–related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B–dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression.

[Image: Diamond nanowires produced by physicist William Gilpin, used only for the purpose of illustration.] As part of some early prep, just putting notes together for a workshop I’ll be leading in Moscow later this summer, I thought I’d link back to this 2014 post by Paul Gilster on Centauri Dreams about “SETI at the … Continue reading "Synthetic at Every Scale"

Hyro Limited group company, Synergy Plus Pty Ltd (“Synergy”) has been named winner of the award for “Leadership in Optimizing IT Infrastructure” in the annual IBM Beacon Awards competition, honoring IBM Business Partners for their ingenuity, innovation, customer satisfaction and outstanding achievements in providing business solutions.

IBM (NYSE:IBM) today announced it has selected Synnex, one of Australia’s largest IT distribution companies, as a new IBM System and Technology Group (STG) distributor. The strategic agreement enables IBM to capture a larger share of the lucrative small and medium business IT marketplace with its IBM Express volume hardware products.

1. Children in US and UK dying from syndrome linked to COVID-19  World Socialist Web Site
2. Rare illness linked to coronavirus kills New York five-year-old  9News
3. Coronavirus US: Andrew Cuomo’s sad announcemet about chld infections  NEWS.com.au
4. Some children with COVID-19 showing symptoms similar to Kawasaki Disease  NEWS10 ABC
5. New fears around 'entirely different chapter' of coronavirus after boy dies from rare illness  Yahoo News Australia
6. View Full coverage on Google News

This article with drawings is in these links https://www.academia.edu/40876793/Synchronizing_moving_GPS_clocks Word, PDF https://pengkuanonphysics.blogspot.com/2019/11/synchronizing-moving-gps-clocks.html PDF 1. Light pulse synchronization Can we synchronize clocks of a moving frame? Let us see Figure 1 where we have stationary frame F1 and moving frame F’2. The 2 clocks in the frame F1 are synchronized with the master clock through a...

This default podcast has been automatically generated by the libsyn system. Feel free to delete it at any time. Welcome to Liberated Syndication, and happy casting

It's not your imagination -- you and your baby really are on the same wavelength. A team of Princeton researchers has now measured baby and adult brain activity moving in sync as they play and interact together.

The Los Angeles Kings have made their home away from Staples Center in the South Bay, an aviation industry hub with an established hockey 'bubble.'

Doesn't really pay for me to be appealing to my captors. They are snarky, and increasingly restless. They pass the long evenings mixing up different flavors of White Claw just for kicks, the way Millennials will.

Letter writers who criticize the president are brushing off accusations from Trump's defenders that they suffer from some kind of insanity.

Watch Gary Varvel's time lapse video of the Tree of Life synagogue shooting.

Hate cannot destroy faith.

strataconf: RT @synergicpartner: We are pleased to announce that we will sponsor next #strataconf in London!!! @strataconf @synergicpartner #BigData ht…

New York Gov. Andrew Cuomo says three children in the state have now died from a possible complication from the coronavirus involving swollen blood vessels and heart problems.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

21 January 2020

Discussions of North African integration have evoked ideas of a shared identity and a common destiny in the region. However, recent attempts to build regional blocs in North Africa have been unsuccessful. This paper examines the benefits of a ‘synergistic’ approach to North African cooperation. 

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Jonathan C. Trinidad
Aug 1, 2012; 11:215-229
Research

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein–coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, Gαq1 null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the Gαq gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in Gαq1 null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

MS Brown
Jul 1, 1980; 21:505-517
Reviews

Margrit Schwarz
Sep 1, 1998; 39:1833-1843
Articles